Human growth hormone (HGH) is a species-specific polypeptide hormone secreted by the pituitary. It is directly implicated in various anti-insulin actions, and indirectly involved in the promotion of growth. Its activity in the latter regard has led to assay of sera for HGH content as an aid in the diagnosis of such diseases as hypopituitary dwarfism (human growth hormone insufficiency) and the form of giganticism known as acromegaly (human growth hormone excess). The use of HGH assay in large-scale screening of potentially afflicted subjects has heretofore been problematic, owing to the fact that HGH levels in the blood are variable, even in normal subjects, as a function of food intake, exercise, and the like. Before reliable diagnosis can be made, it has been necessary in most instances to repeat assays.
The Somatomedins are proteins, probably generated in the liver, and possibly in other organs, as a result of the influence of HGH. In turn, these stimulate increased protein synthesis, cell proliferation and chondrogenesis, the latter leading to increased skeletal growth. Unlike HGH, the blood levels of the Somatomedins are relatively constant, permitting one-time assay for diagnosis. Somatomedin-C is one of a larger group of Somatomedin proteins. Its blood level is regulated by HGH. Somatomedin-C concentrations have been found to correlate well with HGH deficient and HGH excessive humans by radioimmunoassay, when compared to normal populations. Furlanetto, R. W., et al., J. Clin. Invest., 60: 648, 1977. All references cited heretofore and hereafter are incorporated by reference into this application to illuminate the background of the invention.
Because of the relative constancy of Somatomedin-C levels in blood count specimens, blood may be drawn at any time of day, unaffected by food intake and without necessary resort to exercise or other stimulation. Heretofore, to obtain samples for Somatomedin-C assay, it has been necessary to draw on the order of five milliliters of blood, prevent clotting by the addition of Ethylene Diamine Tetraacetate (EDTA), then separate plasma, which is next frozen for shipment to the reference laboratory where the assay is to be conducted. If blood is collected for serum, it must be chilled immediately, centrifuged at low temperature, and kept frozen until the time of assay.
Aside from the general inconvenience of drawing a large blood sample (particularly from children), certain peculiar properties of Somatomedin-C have prevented entirely accurate and efficient assays of Somatomedin-C. Somatomedin-C is "generated" in extracted serum and plasma in a temperature, time, pH and divalent cation dependent process. Blethen, S. L., et al., Proceedings of The Endocrine Society, 61st Annual Meeting, Abstract 592, June 13-15, 1979. The immunoreactive Somatomedin-C concentration in serum may increase as much as three-fold, merely upon standing at room temperature. This can lead to a serious lack of uniformity in collecting and storing sera for purpose of Somatomedin assay. This phenomenon is hereafter referred to as the "exogenous generation" of Somatomedin-C, and is believed to be the result of an enzymatic process acting on a serum macromolecule. Were a chilled blood sample, drawn from a normal patient, to thaw well before assay, inordinately high levels of Somatomedin-C might be found, leading to an incorrect diagnosis of acromegaly. Alternatively, were the same thing to happen in the case of a sample drawn from a subject suffering from undiagnosed hypopituitary dwarfism, the assay of Somatomedin-C might indicate normal levels, owing to Somatomedin generation in the sample following its collection. This problem has led leading workers in the field to suggest that "serum Somatomedin assays may yield anomalous results unless conditions of collection and storage are rigorously standardized". Proceedings, supra.
A need accordingly has existed for methods and means of Somatomedin assay, unattended by the two-fold problems that have characterized past practice: extraction and storage of large volumes of blood, and the relative unreliability of serum assays and resultant occasions for misdiagnosis of HGH content in human patients.